LFA-1 inhibitor and methods of preparation and polymorph thereof

ABSTRACT

Methods of preparation and purification of a compound of Formula I, intermediates thereof, a polymorph thereof, and related compounds are disclosed. Formulations and uses thereof in the treatment of LFA-1 mediated diseases are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/950,807, filed on Jul. 25, 2013, which claims priority from U.S.Provisional Application Ser. No. 61/675,663, filed Jul. 25, 2012, U.S.Provisional Application Ser. No. 61/680,099, filed Aug. 6, 2012, andU.S. Provisional Application Ser. No. 61/729,294, filed Nov. 21, 2012,the disclosures of which are hereby incorporated by reference in theirentireties.

BACKGROUND OF THE INVENTION

The compound of Formula I:

has been found to be an effective inhibitor of LymphocyteFunction-Associated Antigen-1 (LFA-1) interactions with the family ofIntercellular Adhesion Molecules (ICAM), and has desirablepharmacokinetic properties, including rapid systemic clearance. However,improved methods of preparation are useful for providing the compound ofFormula I with increased purity and/or with reduced use of startingmaterials.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a flow diagram showing correlation between different Forms ofFormula I.

FIG. 2 is a flow diagram showing inter-conversion between Forms I, IIIand VI of Formula I.

FIG. 3 is a ternary phase diagram of Formula I in an aqueous acetonesystem.

FIG. 4 is a graphical representation of the X-ray powder diffractionpattern of crystalline Form II.

FIG. 5 is a graphical representation of the optical micrograph ofcrystalline Form II.

FIG. 6 is a graphical representation of the ¹H NMR spectrum ofcrystalline Form II.

FIG. 7 is a graphical representation of the DSC thermogram ofcrystalline Form II.

FIG. 8 is a graphical representation of the TGA thermogram ofcrystalline Form II.

FIG. 9 is a graphical representation of gravimetric moisture sorptioncurve of crystalline Form II.

FIG. 10 is a characterization summary of the Forms of Formula I.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides methods of making a compoundof Formula I:

or a salt thereof. According to the invention, such methods comprise thesteps of performing hydrolysis of a precursor ester with a base underbiphasic conditions where the precursor ester group is acarbon-containing moiety or a silyl-containing moiety; and b) isolatingthe compound of Formula I or a salt thereof. In various embodiments, thebiphasic conditions include aqueous acetone, such as 30% aqueousacetone. In various embodiments, the biphasic conditions change overtime such that a reaction mixture that is biphasic at initiation ofreaction becomes less biphasic or monophasic as the reaction proceeds.

In various embodiments, the base for hydrolysis is sodium hydroxide, forexample, in amounts ranging from about 1.0 to about 1.5 equivalents,preferably about 1.2 equivalents.

In various embodiments, the precursor ester includes an ester R groupwhich is a substituted or unsubstituted group selected from lower alkyl,lower alkenyl, lower alkynyl, cyclo(lower)alkyl, cyclo(lower)alkenyl,aryl, aralkyl, heterocyclyl, and heteroaryl groups. Preferably, theester R group is a benzyl group.

In various embodiments, the invention provides methods of making acompound of Formula I requiring the use of a phase transfer catalyst forperforming base-catalyzed hydrolysis. In various embodiments, the phasetransfer catalyst is a quaternary ammonium salt such astetrabutylammonium hydroxide. Such phase transfer catalysts may bepresent in an amount ranging from about 0.01 equivalents to about 0.5equivalents.

In a second aspect, the invention provides compositions which arereaction mixtures corresponding to the methods of making the compound ofFormula I as described above.

In a third aspect, the invention provides methods of purifying acompound of Formula I by recrystallization. In various embodiments, therecrystallization is performed with aqueous acetone. Accordingly,methods are provided comprising a) obtaining crude compound of Formula Ior a salt thereof and recrystallizing the crude compound with aqueousacetone; and b) isolating the compound of Formula I or a salt thereof byremoval of aqueous acetone. Preferably, the aqueous acetone is about 30%aqueous acetone. In various embodiments, the aqueous acetone is used inan amount of about 7 volumes. Preferably, the method is performed for aperiod of time ranging from about 1 hour to about 48 hours.

In a fourth aspect, the invention provides compositions which arerecrystallization mixtures corresponding to the methods of purifying acompound of Formula I as described above.

In a fifth aspect, the invention provides a compound of Formula Isynthesized according to the methods described herein, or recrystallizedaccording to the methods described herein, or both. Preferably, thecompound is essentially free of methyl ethyl ketone. In variousembodiments, the compound of Formula I has an enantiomeric excess ofgreater than about 96% upon isolation from the reaction mixture forbase-catalyzed hydrolysis and prior to recrystallization. In variousembodiments, the compound of Formula I synthesized and/or recrystallizedaccording to the methods of the invention has an enantiomeric excess ofgreater than about 98%.

In a sixth aspect, the invention provides a compound of Formula Iwherein the compound is polymorph Form II as described herein. Invarious embodiments, the compound polymorph Form II is present in asolid composition with a pharmaceutically acceptable carrier. In variousembodiments, the composition is at least about 50% by weight Form II, oralternatively, less than about 5% by weight Form II. In variousembodiments, the solid composition further comprises one or more solidforms selected from the group consisting of amorphous, Form I, Form III,Form IV, Form V, and Form VI.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION

While selected embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe appended claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs.

As used in the specification and claims, the singular form “a”, “an” and“the” includes plural references unless the context clearly dictatesotherwise.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are suitable for pharmaceutical use, preferably foruse in the tissues of humans and lower animals without undue irritation,allergic response and the like. Pharmaceutically acceptable salts ofamines, carboxylic acids, and other types of compounds, are well knownin the art. For example, S. M. Berge, et al., describe pharmaceuticallyacceptable salts in detail in J Pharmaceutical Sciences, 66: 1-19(1977), incorporated herein by reference. The salts can be prepared insitu during the final isolation and purification of the compounds of theinvention, or separately by reacting a free base or free acid functionwith a suitable reagent, as described generally below. For example, afree base function can be reacted with a suitable acid. Furthermore,where the compounds of the invention carry an acidic moiety, suitablepharmaceutically acceptable salts thereof may, include metal salts suchas alkali metal salts, e. g. sodium or potassium salts; and alkalineearth metal salts, e. g. calcium or magnesium salts. Examples ofpharmaceutically acceptable, nontoxic acid addition salts are salts ofan amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzoate, bisulfate, borate, butyrate, camphorate,camphorsulfonate, citrate, cyclopentanepropionate, digluconate,dodecylsulfate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hernisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate, nicotinate,nitrate, oleate, oxalate, palmitate, pectinate, persulfate,3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate,succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate,undecanoate, valerate salts, and the like. Representative alkali oralkaline earth metal salts include sodium, lithium, potassium, calcium,magnesium, and the like. Further pharmaceutically acceptable saltsinclude, when appropriate, nontoxic ammonium, quaternary ammonium, andamine cations formed by direct reaction with the drug carboxylic acid orby using counterions such as halide, hydroxide, carboxylate, sulfate,phosphate, nitrate, sulfonate and aryl sulfonate.

“Pharmaceutically acceptable carrier” or “pharmaceutically acceptableexcipient” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions of theinvention is contemplated. Supplementary active ingredients can also beincorporated into the compositions.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound described herein. Thus, the term “prodrug” refers to aprecursor of a biologically active compound that is pharmaceuticallyacceptable. A prodrug may be inactive when administered to a subject,i.e. an ester, but is converted in vivo to an active compound, forexample, by hydrolysis to the free carboxylic acid. The prodrug compoundoften offers advantages of solubility, tissue compatibility or delayedrelease in a mammalian organism (see, e.g., Bundgard, H., Design ofProdrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam). A discussion ofprodrugs is provided in Higuchi, T., et al., “Pro-drugs as NovelDelivery Systems,” A.C.S. Symposium Series, Vol. 14, and inBioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which areincorporated in full by reference herein. The term “prodrug” is alsomeant to include any covalently bonded carriers, which release theactive compound in vivo when such prodrug is administered to a mammaliansubject. Prodrugs of an active compound, as described herein, may beprepared by modifying functional groups present in the active compoundin such a way that the modifications are cleaved, either in routinemanipulation or in vivo, to the parent active compound. Prodrugs includecompounds wherein a hydroxy, amino or mercapto group is bonded to anygroup that, when the prodrug of the active compound is administered to amammalian subject, cleaves to form a free hydroxy, free amino or freemercapto group, respectively. Examples of prodrugs include, but are notlimited to, acetate, formate and benzoate derivatives of an alcohol oracetamide, formamide and benzamide derivatives of an amine functionalgroup in the active compound and the like.

“Subject” refers to an animal, such as a mammal, for example a human.The methods described herein can be useful in both human therapeuticsand veterinary applications. In some embodiments, the patient is amammal, and in some embodiments, the patient is human. In variousembodiments, the patient is a non-human animal, such as a dog, cat,rabbit, mouse, rat, cow, horse, pig, or chicken.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having structureswherein a hydrogen is replaced by a deuterium or tritium, or a carbon isreplaced by ¹³C- or ¹⁴C-enriched carbon are within the scope of thisinvention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of atoms that constitutesuch compounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present invention, whether radioactive or not, are encompassedwithin the scope of the present invention.

When ranges are used herein for physical properties, such as molecularweight, or chemical properties, such as chemical formulae, allcombinations and subcombinations of ranges and specific embodimentstherein are intended to be included. The term “about” when referring toa number or a numerical range means that the number or numerical rangereferred to is an approximation within experimental variability (orwithin statistical experimental error), and thus the number or numericalrange may vary from, for example, between 1% and 15% of the statednumber or numerical range. The term “comprising” (and related terms suchas “comprise” or “comprises” or “having” or “including”) includes thoseembodiments, for example, an embodiment of any composition of matter,composition, method, or process, or the like, that “consist of” or“consist essentially of” the described features.

Abbreviations used herein have their conventional meaning within thechemical and biological arts.

Compound of Formula I

The compound of Formula I:

has been found to be an effective inhibitor of LFA-1 interactions withICAM-1. It is a member of a class of directly competitive inhibitors ofLFA-1, binding to ICAM's binding site on LFA-1 directly, and thusexcludes ICAM binding. Directly competitive inhibitors of LFA-1 mayoffer the potential for more effective modulation of the inflammatoryand/or immunologic response than allosteric inhibitors provide becausethese inhibitors occlude the binding site more effectively.Pharmaceutically acceptable salts of Formula I are also included.Additional information regarding the compound of Formula I can be foundin U.S. Pat. No. 8,080,562; US Patent Publication 2009/0298869; USPatent Publication 2011/0092707; U.S. Pat. No. 8,084,047; US2010/0092542; and US Patent Publication 2006/0281739; the entirecontents of each of which are incorporated by reference.

In order to develop clinically useful therapeutics, drug candidates needto be chemically pure enough to administer to a subject and of anacceptable physical form in order to be formulated in pharmaceuticallyacceptable dosage forms. One advantageous route to obtain higher purity,reproducibility of physical form, and stability is to identify one ormore useful crystalline forms. The capacity to exist in differentcrystalline forms is known as polymorphism and is known to occur in manyorganic molecules. These different crystalline forms are known as“polymorphic modifications” or “polymorphs.” While polymorphicmodifications have the same chemical composition, they differ inpacking, geometric arrangement, and other descriptive properties of thecrystalline solid state. As such, these modifications may have differentsolid-state physical properties to affect, for example, the solubility,dissolution rate, bioavailability, chemical and physical stability,flowability, fractability, and compressibility of the compound as wellas the safety and efficacy of drug products based on the compound. Inthe process of preparing a polymorph, further purification, in terms ofgross physical purity or optical purity, may be accomplished as well.

A number of different forms, including crystalline forms, of thecompound of Formula I have been discovered, including the crystallineforms A-E and the amorphous form. While crystallization is oftenperformed on organic compounds, it is not predictable in advance as towhich conditions will provide suitable conditions to lead to formationof a particular crystalline form. Further, it is not predictable as towhich particular crystalline form will provide the necessary mixture ofphysical properties, nonlimiting examples of which are described above,to yield a desirable drug dosage form, once formulated. Additionalinformation regarding the crystalline forms A-E and the amorphous formof the compound of Formula I can be found in U.S. Pat. No. 8,080,562; USPatent Publication 2009/0298869; US Patent Publication 2011/0092707;U.S. Pat. No. 8,084,047; US 2010/0092542; and US Patent Publication2006/0281739; the entire contents of each of which are incorporated byreference.

Methods of Manufacture of the Compound of Formula I

In one embodiment, the compound of Formula I is synthesized as in thefollowing Schemes 1-7. The final product of this synthesis yields thecompound of Formula I as an amorphous solid or as a crystalline formsuch as Forms A-E, or a pharmaceutically acceptable salt, eitherdirectly or indirectly. Variants of this overall route may providesuperior yields, cost of goods, and/or superior chiral purity.

Protecting groups for amino and carboxy groups are known in the art. Forexample, see Greene, Protective Groups in Organic Synthesis, WileyInterscience, 1981, and subsequent editions.

In various embodiments in the subsequent schemes, HATU is used as areagent in amide-bond forming reactions. Alternatively, HATU is notused. In various embodiments, at least one amide-bond forming reactionis performed with thionyl chloride as a reagent in place of HATU. Invarious embodiments, all amide-bond forming reactions are performed withthionyl chloride as a reagent to form acid chlorides.

A first alternative protecting strategy produces compound 5′, aprotected species as shown in Scheme 1. The synthesis begins byreductively aminating 3,5, dichlorobenzaldehyde, compound 1′.Cyclization of compound 2′ provides compound 3′. Protection of the freeamine of compound 3′ as a protected species provides compound 4′. Acarboxylic acid functionality is introduced by treatment of compound 4′with introduction of carbon dioxide, to produce compound 5′. In variousembodiments, the protecting group of compound 4′ is a benzofuranylcarbonyl moiety derived from compound 18′.

In various embodiments, upon scaleup to multikilogram and larger scalereactions, treatment of compound 4′ with strong base (such asn-butyllithium (nBuLi) to generate a lithio species, or lithiumdiisopropyl amide (LDA) to generate the lithio species) is performed inflow mode rather than batchwise reaction due to instability of lithiospecies except at cold temperatures. Flow rates and residence times maybe adjusted to maximize yield.

In various embodiments, 6-hydroxy-1,2,3,4-tetrahydro-isoquinoline(Compound 3″) is used as a starting material for Compound 5′. Thestarting material is chlorinated (×2) for example, withN-chlorosuccinimide. In various embodiments, the chlorination isperformed in the presence of a sulfonic acid. In various embodiments,the sulfonic acid is selected from p-toluenesulfonic acid andmethanesulfonic acid. Following protection of the amino group, thehydroxy group is functionalized, for example, as the triflate ester,which is carbonylated to yield the amino-protected methyl ester.Hydrolysis of the methyl ester yields the amino protected carboxylicacid.

In various embodiments, bromophenylalanine is used as the startingmaterial for a portion of the final molecule as shown in Scheme 2. Thestarting material is protected with an amino protecting group to allowfor introduction of a methyl sulfone functionality in compound 8′.Protecting groups are rearranged by introduction of an orthogonalprotecting group for the carboxylic moiety, followed by deprotection ofthe amino group to provide compound 10′. In various embodiments,expensive or exotic bases are replaced with carbonate base such aspotassium carbonate or calcium carbonate as a reagent.

In various embodiments, 3-methylsulfonylbenzaldehyde is converted intothe 3-methylsulfonylphenylalanine derivative and functionalized to yieldcompound 10 as shown above.

Compounds 5′ and 10′ are joined through amide bond formation followed bydeprotection of the remaining amino group in the presence of thecarboxylic protecting group to yield compound 12′ or a salt thereof,such as the HCL salt.

As an alternative to Scheme 3, compound 10″ is coupled with compound 5′to yield the bromo compound 12″, with subsequent introduction of amethyl sulfone functionality in place of bromine at a later step toproduce compound 19′. Alternatively, instead of a bromine, compound 10″includes X, where X is any halide (Cl, I, Br, F) or a leaving group suchas OTs, OTf, or the like.

The benzofuranyl carbonyl moiety of the compound of Formula I can beprepared using various alternative schemes. In one embodiment, thebenzofuranyl carbonyl moiety is prepared by protecting the hydroxylgroup of compound 13′, reducing the carbonyl of compound 13′ to yieldthe benzofuranyl moiety, followed by carboxylation to yield compound18′.

Scheme 4A

In one embodiment, compound 18′ is prepared from 6-hydroxybenzofuran viathe triflate ester and the 6-carboxy methyl ester as intermediates, asshown in Example 4A.

The benzofuran carboxylic acid 18′ is coupled with compound 12′ (or asalt thereof) by amide bond formation to yield protected compound 19′,as shown in Scheme 5. Amide bond formation is known in the art

As an alternative to Schemes 3-5, compounds 18′ and 5″ may be coupledthrough amide bond formation followed by deprotection of the remainingcarboxylic group to form compound 12″. Amide bond formation betweencompound 12″ and 10′ yields compound 19′ with a protected carboxylicgroup.

As an alternative to Schemes 1-5, compounds 12″ and 10″ may be coupledthrough amide bond formation followed by introduction of a methylsulfone functionality in place of the bromine in converting compound 19″to compound 19′ (similar to Scheme 2). Alternatively, instead of abromine, compound 10″ includes X, where X is any halide (Cl, I, Br, F)or a leaving group such as OTs, OTf, or the like. Compound 12″ can alsobe made using the following scheme:

Final deprotection of compound 19′ to yield the compound of Formula I ora salt thereof is accomplished in a variety of ways. In variousembodiments, the resulting compound of Formula I is provided in higheroptical purity and/or higher overall purity and/or higher overall yield.

In one approach, an ester protecting group is removed with acidcatalyzed hydrolysis. For example, a methyl ester protecting group isremoved with acid catalyzed hydrolysis. Alternatively, a benzyl esterprotecting group is removed with acid, for example HCl in dioxane. Thesolvent for acid-catalyzed hydrolysis may be any industrially availablesolvent such as an aprotic solvent, a protic solvent, a polar solvent, anon-polar solvent, an ionic solvent, or a pressurized gas such assupercritical carbon dioxide. In various embodiments, the solvent is anaprotic solvent such as dioxane or tetrahydrofuran or acetone.Variously, the solvent may be selected from hexane, benzene, toluene,1,4-dioxane, chloroform, diethyl ether, dichloromethane,tetrahydrofuran, ethyl acetate, acetone, dimethylformamide,acetonitrile, dimethyl sulfoxide, n-butanol, isopropanol, n-propanol,ethanol, methanol, water, formic acid, acetic acid, trifluoroaceticacid, and combinations thereof, such as aqueous acetone. The acid may beany acid used for hydrolysis reactions. In various embodiments, the acidis a mineral acid. In various embodiments, the acid is selected fromhydrogen chloride, sulfuric acid, phosphoric acid, and sulfonic acids.In various embodiments, the acid is trifluoroacetic acid. In oneembodiment, the ester may be removed by nucleophilic displacement, suchas using sodium iodide in dimethylsulfoxide.

In one approach, a benzyl ester protecting group is removed withpalladium on carbon. For example, the benzyl ester of compound 19′ isremoved by transfer hydrogenolysis using 10% palladium on carbon, usingformic acid and triethylamine in a 5:1 mixture of methanol:THF, toproduce the compound of Formula I.

In various embodiments, the compound 19′ is a compound of Formula AA. Ageneral strategy to convert a compound of Formula AA is provided by basehydrolysis of the ester to yield the compound of Formula I.

The compound of Formula AA may be reacted with a base in a solvent toaccomplish the base-catalyzed saponification of Formula AA to yield thecompound of Formula I.

The saponification solvent may be any industrially available solventsuch as an aprotic solvent, a protic solvent, a polar solvent, anon-polar solvent, an ionic solvent, or a pressurized gas such assupercritical carbon dioxide. In various embodiments, the solvent is anaprotic solvent such as dioxane or tetrahydrofuran or acetone.Variously, the solvent may be selected from hexane, benzene, toluene,1,4-dioxane, chloroform, diethyl ether, dichloromethane,tetrahydrofuran, ethyl acetate, acetone, dimethylformamide,acetonitrile, dimethyl sulfoxide, n-butanol, isopropanol, n-propanol,ethanol, methanol, water, and combinations thereof. In a preferredembodiment, the solvent is aqueous acetone. The base may be any baseused for saponification reactions. In various embodiments, the base is ahydroxide such as potassium hydroxide or sodium hydroxide or lithiumhydroxide.

In various embodiments, the R group is any carbon containing moiety.Such compounds may be useful as synthetic intermediates of compounds ofFormula I, or as prodrugs of Formula I. Within the group where R is anycarbon containing moiety, R may be selected from lower alkyl, loweralkenyl, lower alkynyl, cyclo(lower)alkyl, cyclo(lower)alkenyl, aryl,aralkyl, heterocyclyl, and heteroaryl, any of which can be substitutedor unsubstituted. In various embodiments, the lower alkyl group ismethyl, ethyl, propyl, isopropyl, butyl, pentyl, isobutyl, t-butyl, orhexyl. In various embodiments, the R group of Formula AA is a benzylgroup. In various embodiments of Formula AA, the carbon-containingmoiety R does not include a benzyl group.

In various embodiments the R group is a silyl-containing moiety suchthat Formula AA is a silyl ester.

In one embodiment, an ester protecting group is removed with basecatalyzed hydrolysis in a homogeneous reaction such as a reaction insolution. For example, a benzyl ester protecting group is removed withNaOH in aqueous dioxane. In one embodiment, a benzyl ester protectinggroup is removed with NaOH in aqueous acetone. In various embodiments ofa homogeneous liquid reaction, NaOH may range from about 0.1N to about2N, such as about 0.5 N, 0.6 N, 0.7 N, 0.8 N, 0.9 N, 1.0 N, 1.1 N, 1.2N, 1.3 N, 1.4 N, or 1.5 N, with all listed concentrations understood tobe “about”.

In one embodiment, an ester protecting group is removed from Compound19′ or Formula AA with base catalyzed hydrolysis in a heterogeneousreaction in the presence of phase transfer catalyst. For example,Compound 19′ or a compound of Formula AA is contacted with phasetransfer catalyst in aqueous acetone. In various embodiments, thereaction occurs in the presence of a solid-liquid interface. In variousembodiments, the reaction occurs in a slurry of solvent and crystallinematerial. In various embodiments, the reaction is biphasic. In variousembodiments, the reaction begins as a biphasic batch reaction andbecomes increasingly homogeneous as the reaction proceeds and startingmaterial is converted to product which remains in solution. In variousembodiments, racemization of starting material is minimized by reducingexposure of unreacted starting material to base through the use ofbiphasic conditions.

In various embodiments, the progress of the reaction is monitored byassessing the level of solid material remaining. In various embodiments,the reaction is deemed to be essentially complete when the reactionmixture is essentially monophasic (i.e. all solids have been dissolvedinto solution).

In various embodiments, base hydrolysis is performed with an amount ofbase ranging from about 0.9 equivalents to about 3 equivalents, such asabout 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 equivalents, all amountsbeing about. In various embodiments, the amount of base ranges fromabout 1.0 to about 1.5 equivalents, such as about 1.2 equivalents. Invarious embodiments, the base is NaOH. In various embodiments, basehydrolysis is performed with NaOH as base in the presence of less than astoichiometric amount of tetrabutylammonium hydroxide.

In various embodiments, the reaction is a batch reaction with a time tocompletion of greater than 0 hours and less than about 24 hours, lessthan about 12 hours, less than about 8 hours, less than about 6 hours,or less than about 4 hours.

In various embodiments, base catalyzed hydrolysis of compound 19′ or thecompound of Formula AA is performed in the presence of a phase transfercatalyst. In various embodiments, the phase transfer catalyst is aquaternary ammonium salt, a phosphonium salt, or a crown ether. Invarious embodiments, the phase transfer catalyst is selected frombenzyltrimethylammonium chloride, hexadecyltributylphosphonium bromide,tetrabutylammonium hydroxide, tetrabutylammonium bromide,methyltrioctylammonium chloride, and tetrabutylammonium chloride. Invarious embodiments, the phase transfer catalyst is tetrabutylammoniumhydroxide. In one embodiment, the amount of phase transfer catalyst isless than a stoichiometric amount. For example, the amount of phasetransfer catalyst is about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 equivalents,all amounts being about.

In further embodiments, the ester protecting group can be removed byother procedures known in the literature including slightly acidic andslightly basic conditions. The ester protecting groups can also beremoved by treatment with ester hydrolyzing enzymes like pig liveresterase, cholesterol esterase, amino esterase, etc. The removal of theester protecting group from Formula AA can further be achieved byapplication of strong acid resins, weak acid resins, strong base resins,or weak base resins.

Upon formation of the compound of Formula I as a crude compound, avariety of isolation and/or purification methods are available. Thecompound of Formula I may be isolated as a crude product throughdistillation or evaporation of solvent from the final deprotection step.Removal of solvent may be through removal to dryness, or through removalof a portion of solvent to yield a solid/liquid mixture which isfiltered and/or washed. Crude compound may be purified by slurrying in asolvent such as methyl ethylketone (MEK), acetonitrile, methylenechloride, or acetone, which solvents may be aqueous or nonaqueous. Thecompound for Formula I may be isolated/purified throughrecrystallization and/or washing with additional solvents. Proceduresfor recrystallization in small and large scales are known in the art.

A partial list of useful solvents for the preparation and purificationof the compound of Formula I includes, for example, water, aliphaticsolvents such as pentane, petroleum ether, and hexane; aromatic solventssuch as toluene and xylene, aliphatic ketones and esters such as methylethyl ketone, acetone, ethyl acetate, isopropyl acetate, and butylacetate, alcohols, such as ethyl alcohol, propyl alcohol, and methylalcohol, acetonitrile, ethers, such as ethyl ether, tert-butyl methylether (TBME), and tetrahydrofuran, alkenes and alkynes, alkenyl estersand alcohols, alkynyl esters and alcohols, and aromatic esters andalcohols. In one embodiment, recrystallization is performed inpharmaceutically acceptable solvent(s). In one embodiment, a usefulsolvent is aqueous acetone.

In various embodiments, recrystallization is performed with from about0.5 volumes to about 15 volumes of recrystallization solvent, forexample from about 5 volumes to about 15 volumes, or for example about1, 2, 3, 4, 5, 6, 7, 8, or 9 volumes. In various embodiments,recrystallization is performed with at least about 10 volumes ofrecrystallization solvent. In various embodiments, recrystallizationprovides one or more crops of crystals, for example 1 crop, 2 crops, 3crops, or more. In various embodiments, recrystallization provides ayield of at least 50%, or at least 60%, or at least 70%, or at least80%, or at least 90% in a first filtration and/or in a combination offiltrations.

In various embodiments, final deprotection and/or recrystallization isperformed in aqueous acetone. Water and acetone are miscible thusallowing for a range from 100%/0% water/acetone to 0%/100% wateracetone. In various embodiments, the ratio of water/acetone is about10/90, 20/80, 30/70, 40/60, 50/50, 60/40, 70/30, 80/20, or 90/10, allamounts being “about”. Preferably, the solvent for final deprotectionand/or recrystallization is about 30% aqueous acetone. In variousembodiments, recrystallization with aqueous aceone provides a yield ofat least 50%, or at least 60%, or at least 70%, or at least 80%, or atleast 90% in the first filtration and/or in a combination offiltrations. In various embodiments, aqueous acetone is used from about0.5 volumes to about 15 volumes, such as about 7 volumes, as providedabove.

In various embodiments, a pH modifier is used during isolation and/orpurification of the compound of Formula I. Without wishing to be boundby theory, it is believed that the solubility of the compound of FormulaI is modified by exposing a salt of the compound of Formula I to acidicconditions such that the carboxylic acid moiety of the compound ofFormula I is protonated, thus making the compound of Formula I moresoluble in organic solvents. In various embodiments, a pH modifier isadded to a composition of crude compound of Formula I to produce a pHless than about 7. In various embodiments, the pH is lowered to lessthan about 5, less than about 4, or less than about 3. In variousembodiments, the pH is within a range of about 1 to about 5. In variousembodiments, the pH is about 2. The pH modifier may be an acid, such asan organic or mineral acid. In various embodiments, the pH modifier ishydrochloric acid. In various embodiments, the pH modifier is dilute HClsolution, such as 4 N HCl, 1 N HCl solution, 0.1 N HCl, or 0.01 N HClsolution. In various embodiments, local pH of less than about 1 isavoided so as to reduce racemization and/or hydrolysis.

In various embodiments, recrystallization is performed at a temperatureabove room temperature. In various embodiments, recrystallization isperformed at a temperature between about 50 C and about 90 C. In variousembodiments, compound of Formula I is dissolved in recrystallizationsolvent at a temperature above room temperature, filtered to removeparticulates, cooled to room temperature or less than room temperaturesuch that crystallization occurs, and filtered to separate crystals andmother liquor.

In various embodiments, recrystallization is performed in a batchprocess with a time to completion of greater than 0 hours and less thanabout 3 days, less than about 2 days, less than about 36 hours, lessthan about 24 hours, less than about 12 hours, less than about 8 hours,less than about 6 hours, or less than about 4 hours.

In various embodiments, recrystallization is performed in a batchprocess on a scale larger than about 10 kilograms, 100 kilograms, onemetric ton, or 10 metric tons, all amounts being about. In variousembodiments, final deprotection and/or recrystallization is performedwith a yield of at least 60%, or at least 70%, or at least 80%, or atleast 90% in the first filtration and/or in a combination offiltrations.

In further embodiments, the compound Formula I can be purified by otherprocedures known in the literature including but not limited to crashingout of a solution, freeze-drying or lyophilization, dialysis, or thelike.

In some of the embodiments of the methods of manufacture of theinvention, the chiral purity of the compound of Formula I as measured bychiral chromatography at 260 nm is greater than about 75%, about 75.5%,about 76%, about 76.5%, about 77%, about 77.5%, about 78%, about 78.5%,about 79%, about 79.5%, about 80%, about 80.5%, about 81%, about 81.5%,about 82%, about 82.5%, about 83%, about 83.5%, about 84%, about 84.5%,about 85%, about 85.5%, about 86%, about 86.5%, about 87%, about 87.5%,about 88%, about 88.5%, about 89%, about 89.5%, about 90%, about 90.5%,about 91.0%, about 91.5%, about 92.0%, about 92.5%, about 93.0%, about93.5%, about 94.0%, about 94.5%, about 95.0%, about 95.5%, about 96.0%,about 96.5%, about 97.0%, about 97.5%, about 98.0%, about 98.5%, about99.0%, about 99.5%, or about 99.9% of the S-enantiomer. In variousembodiments, the chiral purity of the compound of Formula I as measureby chiral chromatography is greater than about 99%. In some embodiments,the chiral purity of the compound of Formula I as measured by chiralchromatography at 260 nm is about 100%.

In some of the embodiments of the methods of manufacture of theinvention, the compound of Formula I has less than about 2.0%, about1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%, about 1.4%, about1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, about0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%,about 0.04%, about 0.03%, about 0.02%, about 0.01%, or about 0.009% ofany one impurity introduced, obtained or produced as a result of thechemical synthesis, as measured by chromatography at 220 nm. In someembodiments, the impurity is a by-product of the synthesis. In variousembodiments, the impurity is a bromine-containing compound. In variousembodiments, the impurity is a mono-chloro compound.

In some of the embodiments of the method of manufacture of theinvention, the compound of Formula I comprises less than about 3.0%,about 2.8%, about 2.6%, about 2.4%, about 2.2%, about 2.1%, about 2.0%,about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%, about 1.4%,about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%,about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%,about 0.1%, or about 0.09% of total impurities introduced, obtained orproduced as a results of the chemical synthesis, as measured bychromatography at 220 nm. In some embodiments the impurities comprise aby-product of the chemical synthesis.

In one embodiment, the product of recrystallization has less than 0.5%,0.4%, 0.3%, 0.2%, or 0.1% non-pharmaceutically acceptable solvent. Invarious embodiments, the product of recrystallization is essentiallyfree of non-pharmaceutically acceptable solvents. In one embodiment, theproduct of recrystallization has less than 0.5%, 0.4%, 0.3%, 0.2%, or0.1% methyl ethyl ketone.

In various embodiments, compounds synthesized according to the inventionmay have various advantages, such as ease of purification, reduced cost,reduced number of synthetic steps, higher overall yields, reducedimpurities, differing impurity profiles, and reduced racemization of thechiral center. In one embodiment, the compound synthesized according tothe invention has an enantiomeric excess (ee) selected from greater thanabout 95% ee, about 96%, about 97%, about 98%, about 99%, and about99.9%. In various embodiments, the compound synthesized according to theinvention has reduced levels of chemical catalyst as an impuritycompared to a compound of Formula I made using palladium as a catalystto remove an ester group to yield the carboxylic acid. For example, invarious embodiments, the compound has less than 100 ppm contaminationwith palladium, or less than 50 ppm, or less than 10 ppm, or less than 1ppm contamination with palladium. In various embodiments, the compoundis essentially free of chemical catalyst.

The anhydrous form of Formula I and five polymorphs, Forms A, B, C, Dand E, have been previously isolated and characterized. See U.S. Pat.No. 8,080,562. Herein, a novel polymorph of Formula I has beenidentified, isolated and fully characterized. These six Forms are nowreferred as Forms I-VI, as shown in Table 1, which summarizes therelationship between the previously assigned and current nomenclature.

TABLE 1 Form Previous assignment I (Channel Hydrate) A II (Monohydrate)— III (Monohydrate) E IV (Monohydrate) C V (Anhydrate) B VI (Hydrate) D

Pharmaceutical Compositions and Formulations and Kits

In various embodiments, the amorphous form or any of the crystallineForms A, B, C, D, or E, or a combination thereof of the compound ofFormula I are administered in pharmaceutical compositions. Thepharmaceutical compositions of the invention comprise pharmaceuticallyacceptable carriers and excipients as well as the amorphous form or anyof the crystalline Forms A, B, C, D, or E, or a combination thereof ofthe compound of Formula I, in order to formulate the composition forappropriate administration to the subject.

In some of the embodiments of the invention, the crystalline formremains in crystalline form in the pharmaceutical composition. In otherembodiments, the amorphous form and/or crystalline form is solubilizedand is no longer crystalline. In the latter case, however, the superiorpurity or other physicochemical properties of the amorphous form and/orcrystalline form contributes to, i.e., for example, ease of handling theform of the compound of Formula I to form the composition, superiorstorage capabilities of crystalline form prior to formulation, bettertherapeutic index, tolerability of the compound of Formula I to thesubject, or decreased side effects of the compound of Formula I. Theamorphous form or crystalline Forms A, B, C, D, or E may be milled toprovide desirable properties for formulation.

The pharmaceutical compositions of the invention may be formulated as agel, cream, lotion, solution, suspension, emulsion, ointment, powder,crystalline forms, spray, aerosol, foam, salve, paste, plaster, paint,microparticle, nanoparticle, or bioadhesive, and may be prepared so asto contain liposomes, micelles and/or microspheres. Oral formulationscan be tablets, capsules, troches, pills, wafers, chewing gums,lozenges, aqueous solutions or suspensions, oily suspensions, syrups,elixirs, or dispersible powders or granules, and the like and may bemade in any way known in the art. Oral formulations may also containsweetening, flavoring, coloring and preservative agents.

The amorphous form or any of the crystalline forms of the compound ofFormula I, or a combination thereof, may be formulated as a sterilesolution or suspension, in suitable vehicles, well known in the art.Suitable formulations and additional carriers and excipients aredescribed in Remington “The Science and Practice of Pharmacy” (20^(th)Ed., Lippincott Williams & Wilkins, Baltimore Md.), the teachings ofwhich are incorporated by reference in their entirety herein.

The formulations of the invention can further include otherpharmacological active ingredients as far as they do not contradict thepurpose of the present invention. In a combination of plural activeingredients, their respective contents may be suitably increased ordecreased in consideration of their effects and safety.

The invention also provides kits. The kits include a compound of theinvention in suitable packaging, and written material that can includeinstructions for use, discussion of clinical studies, listing of sideeffects, and the like. The kit may further contain another therapeuticagent that is co-administered with the compound of Formula I, includingthe amorphous form or any of the crystalline Forms of the compound ofFormula I or a combination thereof. In some embodiments, the therapeuticagent and the amorphous form or any of the crystalline Forms of thecompound of Formula I or a combination thereof are provided as separatecompositions in separate containers within the kit. In some embodiments,the therapeutic agent and the amorphous form or any of the crystallineforms of the compound of Formula I or a combination thereof are providedas a single composition within a container in the kit. Suitablepackaging and additional articles for use (e.g., measuring cup forliquid preparations, foil wrapping to minimize exposure to air,dispensers, and the like) are known in the art and may be included inthe kit.

Additional information regarding pharmaceutical compositions,formulations, and kits can be found in U.S. Pat. No. 8,080,562; USPatent Publication 2009/0298869; US Patent Publication 2011/0092707;U.S. Pat. No. 8,084,047; US 2010/0092542; and US Patent Publication2006/0281739; the entire contents of each of which are incorporated byreference.

Methods of Use

Not intending to limit methods of use by a single mechanism of action,methods disclosed herein involve the inhibition of initiation andprogression of inflammation related disease by inhibiting theinteraction between LFA-1 and ICAM-1 by administering the compound ofFormula I, including the amorphous form or any of the crystalline FormsA, B, C, D, or E, or a combination thereof, of the compound of FormulaI. In some embodiments, such methods provide anti-inflammatory effectsin-vitro and in-vivo, and are useful in the treatment of inflammationmediated diseases and/or the investigation of disease mechanisms.

In particular, the amorphous form or any of the crystalline Forms A, B,C, D, or E, or a combination thereof, of the compound of Formula I canmodulate inflammation mediated by leukocytes. The amorphous form or anyof the crystalline Forms A, B, C, D, or E, or a combination thereof, ofthe compound of Formula I can be used as a therapeutic agent in anydisorder in which antibodies to LFA-1 are shown to be effective. In oneembodiment of the invention, a subject is administered the amorphousform or any of the crystalline Forms A, B, C, D, or E, or a combinationthereof, of the compound of Formula I to modulate inflammationassociated with ocular inflammation. Another embodiment of the methods,a subject with inflammation associated with dry eye syndrome isadministered the amorphous form or any of the crystalline Forms A, B, C,D, or E, or a combination thereof, of the compound of Formula I.

Administration of a pharmaceutical composition comprising the compoundof Formula I, including the amorphous form or any of crystalline FormsA, B, C, D, or E, or a combination thereof, of the compound of Formula Imay be by any suitable means. In some embodiments, a pharmaceuticalcomposition comprising the amorphous form or any of crystalline Forms A,B, C, D, or E, or a combination thereof, of the compound of Formula I,is administered by oral, transdermal, by injection, slow releaseintraocular implantation, or aerosol administration.

Additional information regarding uses of the compound of Formula I canbe found in U.S. Pat. No. 8,080,562; US Patent Publication 2009/0298869;US Patent Publication 2011/0092707; U.S. Pat. No. 8,084,047; US2010/0092542; and US Patent Publication 2006/0281739; the entirecontents of each of which are incorporated by reference. Additionalinformation regarding administration of the compound of Formula I can befound in U.S. Pat. No. 8,080,562; US Patent Publication 2009/0298869; USPatent Publication 2011/0092707; U.S. Pat. No. 8,084,047; US2010/0092542; and US Patent Publication 2006/0281739; the entirecontents of each of which are incorporated by reference.

EXAMPLES Example 1

Reductively aminating 3,5-dichlorobenzaldehyde, compound 1, with1-chloro-2-aminoethane and sodium cyanoborohydride provided 35% yield ofcompound 2. Cyclization of compound 2 using aluminum chloride catalysisand ammonium chloride at 185° C. provided compound 3 in 91% yield.Protection of the free amine of compound 3 as the trityl protectedspecies afforded compound 4 in 89% yield. A carboxylic acidfunctionality was introduced by treatment of compound 4 withn-butyllithium (nBuLi) and tetramethylethylenediamine (TMEDA), withsubsequent introduction of carbon dioxide, to produce trityl protectedcompound 5 in 75% yield.

Example 1A

To a glass reactor was charged 3,5-dichlorobenzaldehyde. Absoluteethanol was added to the batch slowly (this addition is mildlyexothermic) and agitation started. 2,2-Diethoxyethylamine (1.03 equiv)was slowly added to the batch, keeping the batch temperature at 20-78°C. The batch was then heated to 76-78° C. for 2 h. GC-MS analysisindicated reaction completion (starting material <1%). The batch wascooled to ambient temperature for work-up. The batch was concentrated invacuo to a residue and azeotroped with heptanes (×2). The residue wascooled and held at 0-5° C. for 12 h to form a suspension. The solidswere collected by filtration and the cake was washed with cold (0-5° C.)heptanes, and dried under hot nitrogen (45-50° C.) to afford Compound 2′as a white solid (94% yield).

To a glass reactor was charged concentrated 95-98% sulfuric acid (25.9equiv). The batch was heated to 120-125° C. and a solution of Compound2′ in CH₂Cl₂ was added slowly over 1 h, keeping the batch temperaturebetween 120-125° C. The batch was then stirred at 120-125° C. for 6 h.The batch was cooled to <50° C. To a glass reactor was charged DI waterand the batch temperature was adjusted to 0-5° C. The reaction mixturewas slowly transferred, keeping the batch temperature between 0-50° C.DI water was used to aid the transfer. To the batch was added Dicalite4200. The batch was filtered through a pad of Dicalite 4200. To thefiltrate was added 50% aqueous sodium hydroxide solution slowly over 3h, keeping the batch temperature between 0-50° C. to adjust the pH to12. The resulting suspension was stirred at 45-50° C. for 2 h and thesolids were collected by filtration. The filter cake was slurried in DIwater at 30-35° C. for 1 h. The batch was filtered. The cake was washedwith heptanes and dried in vacuum oven at 45-50° C. for 22 h to givecrude compound 2″ as a tan solid (75% yield), which was further purifiedby recrystallization.

To a reactor was added platinum dioxide (0.012 equiv), Compound 2″, andMeOH (10 vol) and the suspension was stirred at room temperature underargon for 10 minutes. The reaction mixture was inerted with argon threetimes and then stirred under 125 psi of hydrogen at room temperature for25 hours. HPLC analysis indicated complete reaction with less than 1% ofthe starting material remaining After standing, the supernatant wasdecanted from the solids (catalyst) by vacuum. To the solids was addedmethanol and the slurry was mixed under nitrogen. The solids wereallowed to settle on the bottom over several hours. The supernatant wasdecanted from the solids by vacuum. The combined supernatants werefiltered through Celite under a blanket of nitrogen and the filter padwas washed with MeOH (×2). The combined filtrate and washes wereconcentrated to dryness. The residue was slurried in MTBE. The mixturewas treated with 3 M HCl while maintaining the temperature <40° C.resulting in the formation of a heavy precipitate. The mixture wasstirred at 35-40° C. for 60 to 90 minutes. The batch was cooled to 0-5°C., stirred for 60 to 90 minutes and then filtered. The filter cake waswashed with cold DI water (×2) followed by a displacement wash with MTBE(×2). The filter cake was dried under reduced pressure to affordCompound 3 Hydrochloride Salt (86% yield). The hydrogenation catalystcan be recovered and re-used.

Compound 3 and trityl chloride were added to the reaction flask. DCM (10vol) was added to the reactor and agitation was started to form slurry.The reaction mixture was cooled to 10-15° C. N,N-Diisopropylethylamine(2.5 equiv) was slowly added to the reaction mixture, maintaining thetemperature at 15-25° C. during the addition. Once addition wascomplete, the batch was stirred at 15 to 25° C. for a minimum of 60minutes. The reaction was assayed by HPLC by diluting a sample withacetonitrile and then injecting it on the HPLC. The first assay after 30minutes indicated that the reaction was complete with <1% of startingmaterial observed by HPLC analysis. The reaction mixture was dilutedwith DI water (5 vol). The reaction mixture was stirred for 5 minutesafter which it was transferred into a separation funnel and the phaseswere allowed to separate. The DCM layer was washed with DI water (5 vol)by stirring for 5 minutes and then allowing the phases to separate. TheDCM layer was washed with brine (5 vol) by stirring for 5 minutes andthen allowing the phases to separate. The DCM layer was dried overmagnesium sulfate, filtered and the filter cake was washed with DCM(×2). The combined filtrate and washes were concentrated to a residuethat was azeotroped with EtOAc (×2). The residue was suspended in EtOAcand stirred for 1 hour in a 40° C. water bath. The resulting slurry wascooled to 0-5° C. for 1 hour and then filtered. The filter cake waswashed twice with EtOAc and then dried under reduced pressure to affordCompound 4.

Example 1B

To 1,2,3,4-tetrahydro-6-hydroxy-isoqinoline in acetonitrile was addedp-toluenesulfonic acid and N-chlorosuccinimide. The suspension wascooled to ambient temperature, and the product isolated by filtrationfor a yield of approximately 61% with purity greater than 95%. Theisolated TsOH salt was recrystallized until purity was greater than99.7%. To one equivalent of the TsOH salt suspended in methanol wasadded 2M sodium carbonate (0.55 eq.) and 1.2 eq. of Boc anhydride. Thesuspension was stirred at room temperature overnight. The reaction wasmonitored by HPLC. Upon completion, the mixture was cooled to below 10°C., water was added, and the Boc-protected dichloro compound wasisolated by filtraton. The product was washed and dried at 40° C. for ayield of 95% and purity of >97%. The Boc-protected dichloro compound wassuspended in dichloromethane (10 volumes) and pyridine (5 volumes) wasadded. The mixture was cooled to below 2° C., and triflic anhydride(1.25 eq) was added. The mixture was stirred at 0-2° C. for 10 minutes,and then poured into 10 volumes of 6% aqueous sodium hydrogen carbonatesolution. After washing with dichloromethane, the organic phases werecombined and dried over magnesium sulphate. Following purification, theproduct (Compound 4′) was obtained in 90% yield and >98% purity.Compound 4′ was dissolved in dimethylformamide and methanol at roomtemperature. Diisopropylamine (4 eq) was added. Under CO atmosphere,1,3-bis(diphenylphosphino)propane (0.1 eq) and palladium acetate (0.1eq) was added. The reaction was heated to reflux, and monitored by HPLC.Upon near completion, the mixture was cooled to ambient temperature.Workup with water, ethyl acetate, and brine yielded Compound 4″, whichwas used without further purification. Compound 4″ was dissolved inmethanol and 2.4 M sodium hydroxide (10 volumes each) and refluxed. Themixture was cooled to ambient temperature, and toluene was added.Following aqueous workup, the pH was adjusted to 2.3 with 3Mhydrochloric acid, and crude product was isolated by filtration in 53%yield with greater than 80% purity.

Example 2

t-Butylcarbamate (Boc) protection of the amino group ofbromophenylalanine was accomplished, using sodium bicarbonate (3equivalents), t-butyl dicarbonate (Boc₂O, 1.1 equivalent) in dioxane andwater, to obtain compound 7 in 98% yield. A methyl sulfone functionalitywas introduced by treating the bromo compound 7 with copper iodide (0.4equivalents), cesium carbonate (0.5 equivalents), L-proline (0.8equivalents), and the sodium salt of methanesulfinic acid (3.9equivalents) in dimethylsulfoxide (DMSO) at 95-100° C. for a total of 9hours, with two further additions of copper iodide (0.2 equivalents) andL-proline (0.4 equivalents) during that period. Compound 8 was isolatedin 96% yield. The carboxylic acid of compound 8 was converted to thebenzyl ester, compound 9, in 99% yield, using benzyl alcohol (1.1equivalent), dimethylaminopyridine (DMAP, 0.1 equivalent) andN-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC, 1.0 equivalent). Theamino group of compound 9 is deprotected by adding a 4N solution of HClin dioxane to compound 9 at 0° C. in methylene chloride. The HCl salt ofthe free amino species, compound 10 was isolated in 94% yield.

Example 2A

Example 2 was repeated with potassium carbonate in place of cesiumcarbonate.

Example 2B

Boc-protected bromophenylalanine (Compound 7) (100 g) was dissolved inDMSO (400 mL) with stirring and degassing with argon. Sodium methanesulfinate (98 g), copper iodide (28.7 g), potassium carbonate (40 g) andL-proline (26.75 g) were added at 28-30° C. Reaction was heated to about87° C. for about 17-19 hours. Reaction was cooled and quenched withcrushed ice, stirred for 30-40 minutes, and the pH was adjusted fromabout 12 to about 3-4 with citric acid (350 g). Quenched reactionmixture was filtered, extracted with dichloromethane ×3, washed withammonium chloride solution, washed with sodium bisulphite solution, andwashed with brine. Crude product in dichloromethane was concentrated invacuo until moisture content was below about 0.5%, and used in next stepwithout further isolation. Crude compound 8 in dichloromethane wascharged with benzyl alcohol and DMPA with stirring under nitrogen.Reaction cooled to 0-5° C. EDC-HCL (1.03 equiv) added with stirring for30 minutes. Upon completion of reaction by TLC and HPLC, the reactionwas quenched with sodium bicarbonate solution, the organic layer wasseparated, and the aqueous layer was extracted with dichloromethane. Theorganic layer was washed with citric acid solution, and combined organiclayers were washed with brine solution. Dichloromethane was removed at45-50° C., and the concentrate was used for next step without furtherisolation. The amino group of compound 9 was deprotected by adding a 4Nsolution of HCl in dioxane to compound 9 at 10-15° C. in methylenechloride. The HCl salt of the free amino species, compound 10 wasisolated by filtration from diethyl ether. Isolation of compound 10 wasperformed through recrystallization using adimethylformamide/dichloromethane solvent system.

Example 3

Compound 5 was treated with triethylamine (TEA, 5 equivalents) and2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU, 1.25 equivalents) for 10 minutes indimethylformamide (DMF), and then compound 10 was added to the solution.After stirring at room temperature for 18 hours, the product, compound11 was isolated in 70% yield. Removal of the trityl protecting group wasaccomplished by treating compound 11, with HCl in dioxane (4 N, excess)at room temperature for 2 hours, diethyl ether added, and the solidproduct, compound 12, was isolated by filtration in 95% yield. Thecompound 12 exists in both amorphous and crystalline form and can beisolated in either form.

Example 3A

Compound 5 was dissolved in isopropyl acetate and cooled to 20 to 25° C.Thionyl chloride was added, with cooling to 10 to 15° C., andN-methylmorpholine was added slowly. The reaction was monitored by HPLC.Compound 10, water, and isopropyl acetate were stirred at 15 to 20° C.until a solution was achieved. N-methylmorpholine was added followed byaddition of the Compound 5 reaction mixture (acid chloride of Compound5). The reaction was monitored by HPLC. Upon completion, the biphasiclayers were allowed to settle, and the aqueous layer was removed. Theupper organic layer was extracted with water, and the remaining organiclayer was distilled under vacuum. Dioxane and IpAc were added withfurther distillation. Once dry, 4N anhydrous HCl in dioxane was added.The mixture was stirred at 20 to 25° C. for 12 hours, and checked forcomplete deprotection by HPLC. Once complete, the thick slurry wasfiltered, washed with IPAc and dried under vacuum at 45 to 55° C. Yieldof Compound 12 was 88%.

Example 4

The benzofuranyl carbonyl moiety of the compound of Formula I wasprepared using various schemes, (Schemes E4, E4A, and E4B).

The benzofuranyl carbonyl moiety was prepared by protecting the hydroxylgroup of compound 13 by reacting with tert-butyldimethylsilyl chloride(1.0 equivalents) and triethylamine (TEA, 1.1 equivalents) in acetone,to give compound 14 in 79% yield. A solution of compound 14 in methanolwas then treated with sodium borohydride (1.0 equivalent) at roomtemperature overnight. The reaction was quenched with an addition ofacetone, stirred at room temperature for a further 2.5 hours, aqueousHCl (4N) was added with the temperature controlled to below 28° C.,tetrahydrofuran (THF) was added, and the solution stirred overnightunder argon and in the absence of light. The product, compound 15, wasisolated quantitatively by extraction into methylene chloride,concentrated at low heat, and used without further purification. Thetriflate ester, compound 16, was produced in 69% yield from compound 15by reacting it with N-phenyl-bis(trifluoromethanesulfonimide) (1.0equivalent) in methylene chloride for 72 hours. Compound 16 in a mixtureof DMF, methanol, and triethylamine, was added to a prepared solution ofpalladium acetate, 1,3-Bis(diphenylphosphino)propane (dppp), DMF andmethanol in an autoclave. Carbon monoxide was charged into the autoclaveto a pressure of 8 bar, and the reaction mixture was heated at 70° C.for 6 hours. After workup, compound 17 was isolated in 91% yield.Lithium hydroxide (4 equivalents) in methanol and water was used tohydrolyze the ester and permit the isolation of compound 18′ in 97%yield.

Example 4A

Example 4 was repeated with triflic anhydride and sodium hydroxide asreagents for the ester hydrolysis.

Compound 15 (6-Hydroxybenzofuran) was stirred in dichloromethane anddiisopropylethylamine. Triflic anhydride (1.2 eq.) was added, keepingthe temperature below 20 C. The reaction was monitored by HPLC. Thereaction was quenched with methanol, solvent was removed with vacuum,and the crude residue of Compound 16 was used without furtherpurification. Compound 16 as crude residue was dissolved in 4 volumes ofdimethylformamide and 2 volumes methanol. To the solution was added 0.02eq. of palladium acetate, 0.02 eq. of dppp, and CO under pressure. Thereaction was monitored by HPLC. Following workup, Compound 17 wasisolated as a crude oily residue without further purification. Theresidue of compound 17 was dissolved in methanol (5 volumes) and 1volume of sodium hydroxide (27.65%) was added. The mixture was heated to40 C until full conversion of HPLC. The mixture was cooled to ambienttemperature and 3 volumes of water were added. The pH was adjusted toabout 2 with 3M hydrochloric acid. The suspension was filtered, washedwith water, and dried to give Compound 18′ in about 75% overall yieldwith purity >99.5%.

Example 4B

Diethyl 2-(1,3-dioxolan-2-yl)ethylphosphonate, compound 1″, was preparedfrom 2-(2-bromoethyl)-1,3-dioxolane by the addition of triethylphosphate. After removal of ethyl bromide through distillation at 210°C. the crude reaction mixture was cooled and then by way of vacuumdistillation, compound 1″ was collected as a colorless oil in 94% yield.

In the next step, n-butyllithium (2.15 equivalents) in hexane was cooledto −70° C. and diisopropylamine (2.25 equivalents) was added whilekeeping the temperature below −60° C. Compound 1″ (1 equivalent)dissolved in tetrahydrofuran (THF) was added over 30 min at −70° C.After 10 min, diethyl carbonate (1.05 equivalents) dissolved in THF wasadded over 30 min keeping the reaction temperature below −60° C. Afterstirring for one hour at −60° C., the reaction was allowed to warm to15° C. and furan-2-carbaldehyde (1.3 equivalents) dissolved in THF wasadded. After stirring for 20 hrs at room temperature, the reaction wasrotary evaporated to dryness to yield ethyl2-((1,3-dioxolan2-yl)methyl-3-(furan-2-yl)acrylate, which was useddirectly in the next reaction.

The crude compound (1 equivalent) was dissolved in ethanol and added toa mixture of water and phosphoric acid (85%, 15 equivalents) over 30 minwhile keeping the temperature below 50° C. After stirring for 20 hrs atroom temperature, another 200 ml of phosphoric acid (85%) was added andthe mixture was heated to 50° C. for an additional two hrs. Afterremoval of ethanol by rotary evaporation, the material was extractedwith toluene, washed with water, dried with sodium sulfate, treated withcharcoal, filtered and dried down to an oil. This oil was distilled toafford ethyl benzofuran-6-carboxylate, compound 6″, (bp 111-114.5° C.)which crystallized on standing. Compound 6″ was recovered at 57% yieldbased on compound 1″.

Compound 6″ (875 mmol) was dissolved in methanol and tetrahydrofuran(THF). Sodium hydroxide (4 M, 3 equivalents) was added and the reactionwas stirred overnight. After concentration via rotary evaporation, theaqueous solution was extracted with methyl tert-butyl ether (MTBE),acidified to pH 2 with the addition of hydrochloric acid (HCl) andcooled resulting in fine crystals of benzofuran-6-carboxylic acid, i.e.,compound 18′. Compound 18′ was isolated, washed with water and dried toa final yield of 97% yield.

Example 5

The benzofuran carboxylic acid 18′ was treated with oxalyl chloride (1.2equivalents) and a catalytic amount of DMF, stirring for 5.5 hours untila clear solution was obtained. The solvent was removed under reducedpressure and the acid chloride of compound 18′ was stored under argonuntil use, on the next day. The acid chloride, in methylene chloride wasadded slowly to a methylene chloride solution of the compound of Formula12 and diisopropylethylamine (DIPEA) which was cooled to 0-5° C. Thereaction was not permitted to rise above 5° C., and after completion ofaddition, was stirred at 5° C. for a further 0.5 hour. Upon aqueousworkup and extraction with methylene chloride, the product, compound 19,was isolated in quantitative yield.

The benzyl ester of compound 19 was removed by transfer hydrogenolysisusing 10% palladium on carbon, using formic acid and triethylamine in a5:1 mixture of methanol:THF, to produce the compound of Formula I in 95%yield.

A final step of slurrying in methyl ethylketone (MEK) produced Form A ofthe compound of Formula I. The product was washed with water to removeresidual MEK. Alternatively, the product of the hydrogenolysis step wasslurried in acetonitrile to yield Form A of the compound of Formula I.

Taking the compound of Formula I directly as the crude reaction productafter transfer hydrogenolysis, and reconcentrating down from a solutionin methylene chloride, the amorphous form of the compound of Formula Iwas obtained in 97% purity.

Example 6

An alternative protection strategy was performed in Scheme E6.

Boc-protection was used for the ring nitrogen in the intermediates 21and 22. Compound 5 was deprotected with HCl in dioxane to producecompound 23 in better than 97% yield. Boc-protection was introduced,using di-tert-butyl dicarbonate (1.1 equivalent), and compound 21 wasobtained in better than 95% yield. Compound 10 was coupled with compound21 to obtain compound 22, using HATU and triethylamine in DMF. Theproduct, compound 22, was obtained in quantitative yield, and greaterthan 90% purity. Deprotection with HCl yielded the compound of Formula12 in 97.4% yield.

Transfer hydrogenolysis of compound 19 produced the compound of FormulaI with optical purity of 98.5% (S) enantiomer compared to 79-94.5% (S)enantiomer optical purity obtained by hydrolysis of the correspondingmethyl ester.

Example 6A

Example 6 was repeated with thionyl chloride to form the acid chloridefor amide bond coupling in place of HATU.

Example 7

An alternate strategy to convert compound 19 into Formula I wasperformed by base hydrolysis of the benzyl ester (compound 19).

Compound 19 ((S)-benzyl2-(2-(benzofuran-6carbonyl)-5,7-dichloro-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)-3-(3-methylsulfonyl)phenyl)propanoate)(70.9 mmol) was dissolved in dioxane and water was added. This solutionwas cooled to 8° C. Over 45 minutes, NaOH (0.5 M) was added to 68.0mmol. After stirring for 2 hrs, the dioxane was removed by rotaryevaporation. The aqueous solution was extracted twice with toluene toremove unreacted starting material. Ethyl acetate was added to theaqueous layer and with vigorous stirring the aqueous layer was acidifiedto pH 2 with HCl (4 M aqueous). After stirring, the layers wereseparated and the aqueous layer was extracted with ethyl acetate. Thecombined ethyl acetate fractions were washed with brine, dried withsodium sulfate and evaporated to dryness resulting in a foam which was95% pure by HPLC and had a 94.8% ee. This foam was dissolved in methylethylketone (MEK) and was seeded with crystals (99% pure, 99% ee) whichresulted in thick crystallization. After stirring for 24 hr, thesuspension was filtered and washed with water and dried under vacuum.The yield of Formula I was 77% with a purity of 98.9% and 97.9% eeoptical purity. An additional crop of Formula I (>98% pure) was obtainedthrough concentration of the mother liquor.

Example 8

An alternative coupling, deprotection, and purification process wasperformed.

Compound 18′ was dissolved in isopropyl acetate and the temperatureadjusted to 20 to 25° C. Thionyl chloride was added, and the temperaturewas adjusted to 10 to 15° C. N-methylmorpholine was added. The reactionwas monitored by HPLC. Compound 12 was dissolved in water, methyl ethylketone, and N-methylmorpholine, and this mixture was cooled to 15 to 20°C. The acid chloride solution of Compound 18′ was added slowly andstirred for 30 minutes. The reaction was monitored by HPLC. Seeds ofCompound 19 were added, and the mixture was stirred for 1 hour, and thena portion of the organic solvents were distilled under vacuum. Themixture was cooled to 20 to 25° C. and stirred for 2 hours, andfiltered. The filter cake was washed with isopropyl acetate, and thenthe filter cake was slurried in water, filtered, washed with water, anddried at 40° C. under vacuum for a yield of 90%.

Compound 19 was mixed with acetone, water, and tetrabutylammoniumhydroxide (TBAH) and stirred at 18 to 22° C. until a solution wasachieved. 2N sodium hydroxide was added slowly over 1 hour, and stirredat 25° C. until HPLC indicated that the reaction was complete. Themixture was distilled under vacuum at 30 to 35° C. to remove acetone.The resulting solution was cooled to 10° C. and 4N aq HCl was addedmaintaining a temperature of less than 15° C., to pH ˜2. The suspensionthat forms was stirred for about an hour, filtered, and the filter cakewas washed with water. The wet cake was suspended in acetone and water(about 2/1) and warmed to 40 to 45° C. to effect solution. The solutionwas filtered through a 10 micron filter. The mixture was cooled to 18 to22° C. Seeds were added and the mixture was stirred for 12 hours. Theproduct was collected by filtration, and washed with 30% aqueousacetone, and dried under vacuum at 45 to 55° C. for a yield of 88%.

Example 9

Crude compound of Formula I was recrystallized in 10 volumes of methylethyl ketone with stirring for 3 days to yield purified compound ofFormula I in 60-65% yield.

Example 10

Crude compound of Formula I was recrystallized in 30% aqueous acetonefollowed by one volume water over 24-36 hours to yield purified compoundof Formula I in 73-77% yield.

Example 10A

Crude compound of Formula I is recrystallized in 30% aqueous acetonefollowed by one volume water over 24-36 hours to yield purified compoundof Formula I in 80-90% yield with multiple filtrations. The obtainedcompound of Formula I has no detectable residue of methyl ethyl ketone.

Example 11 Crystalline Form II

Small Scale Synthesis

Approximately 50 mg of crystalline Form I was dissolved in acetone (2.5mL) at 50° C. The solution was polish filtered into a preheatedcontainer. Anti-solvent, n-heptane was added and the mixture was placedin a refrigerator at about 5° C. The resulting solid was filtered anddried under vacuum.

Scale Up Synthesis

Approximately 320 mg of Form I was dissolved in acetone (15 mL). Thesolution was polish filtered into a preheated vial. Then n-heptane (10mL) was added and the mixture was refrigerated for 30 min. The cooledsolution was seeded with Form II material and allowed to equilibrate for12 h at 5° C. The resulting solid was filtered and dried overnight undervacuum.

The ¹H-Nuclear Magnetic Resonance spectrum of crystalline Form II shownin FIG. 6 is consistent with structure of compound and contains 0.2 wt %acetone. The crystalline Form II comprises a powder x-ray diffractionpattern as shown in FIG. 4 with an acicular morphology as indicated inFIG. 5. DSC analysis of Form II shows a small endotherm at 37.8° C.,likely due to loss of acetone and/or water, and a melting transition at155.5° C. as presented in FIG. 7. The thermal events assigned in the DSCthermogram of Form II are consistent with the observations from hotstage microscopy analysis of the polymorph.

A thermogravimetric analysis graph of crystalline Form II is shown inFIG. 8. A mass loss of 1.5 wt %, attributed to liberation of waterduring the melt, followed by onset of decomposition at 260° C. isobserved. Gravimetric moisture sorption analysis reveals that Form II ismoderately hygroscopic, absorbing 3.0 wt % water at 60% relativehumidity and 3.4 wt % water at 90% relative humidity. The water contentof the Form II under 40% relative humidity is 2.8 wt %, which is closeto the theoretical water content of a mono-hydrate of the compound (2.9wt %). The water content of Form II by Karl Fisher titration is 3.2 wt%, again in-line with a monohydrate of compound.

While selected embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method of purifying a compound of Formula I:

or a salt thereof, comprising the steps: a) obtaining crude compound ofFormula I or a salt thereof and recrystallizing said crude compound withaqueous acetone; and b) isolating said compound of Formula I or a saltthereof by removal of aqueous acetone.
 2. The method of claim 1, whereinsaid aqueous acetone is used in an amount of about 7 volumes.
 3. Themethod of claim 1, wherein said aqueous acetone is about 30% aqueousacetone.
 4. The method of claim 1, further comprising addition of a pHmodifier to produce a pH of less than about
 5. 5. The method of claim 4,wherein said method produces a pH between about 1 and about
 5. 6. Themethod of claim 4, wherein said pH modifier is selected from an organicor mineral acid.
 7. The method of claim 4, wherein said pH modifier ishydrochloric acid.
 8. The method of claim 1, wherein said removal ofaqueous acetone is by filtration.
 9. The method of claim 1, wherein saidmethod is performed for a period of time ranging from about 1 hour toabout 48 hours.